PcrA (plasmid-copy-reduced A) is an essential protein present in Gram-positive bacteria, including important human pathogens such as Staphylococcus aureus, Streptococcus pneumonia, Clostridium perfringens, Listeria monocytogenes, Bacillus anthracis, etc. PcrA was discovered in S. aureus as a helicase required for the rolling-circle (RC) replication of plasmid pT181. PcrA is related to the Escherichia coli UvrD and Rep helicases and is known to be essential for UV DNA repair in Bacillus subtilis. In B. subtilis, suppressors of pcrA knockout map in the recombination genes recFOR and conditional knockouts of pcrA are hyperrecombinogenic, suggesting an essential role for PcrA in the regulation of RecA-mediated DNA recombination. We have characterized the PcrA protein of S. aureus and shown that it has DNA binding, ATPase, helicase and plasmid DNA unwinding activities. We have also shown that PcrA inhibits RecA-mediated DNA strand exchange in vitro by displacing RecA bound to the DNA. Using PcrA mutants, our preliminary studies suggest that the ATPase and helicase activities of PcrA may not be essential for its antagonistic effects on RecA in vitro. Although the biochemical properties of PcrA from several Gram-positive bacteria have been characterized and structural information is available on Bacillus stearothermophilus PcrA (PcrABst), the biochemical activities and functions of PcrA that are essential for the growth of S. aureus and other Gram-positive bacteria remain unknown. Using genetic and biochemical approaches, this study aims to investigate the biological functions of PcrA that make it an essential protein in S. aureus. In Aim 1, we will puriy a few site-directed mutants of S. aureus PcrA that we have generated based on the known three-dimensional structure of the highly-related PcrABst, and characterize their biochemical activities. We will then create a conditional knockout of S. aureus pcrA and carry out complementation studies using PcrA mutants defective in DNA binding, ATPase, helicase and RecA displacement/inhibition to identify the activities of PcrA important for the growth and viability of S. aureus and the regulation of RecA function in vivo. In Aim 2, we will identify the possible role of PcrA in UV DNA repair and the SOS response, which is known to promote the horizontal transfer of virulence genes and pathogenicity islands in S. aureus. The SOS response is induced by UV light and agents such as antibiotics, and requires the assembly of RecA filaments. We will investigate whether the helicase activity of PcrA is required for UV DNA repair and if PcrA levels increase during the SOS response and regulate RecA functions. Our studies should identify the biochemical activities of PcrA that make it an essential protein in S. aureus. Since PcrA is a conserved protein, the knowledge gained from these studies could be used in the future for the development of drugs against S. aureus and other Gram-positive human pathogens. PUBLIC HEALTH RELEVANCE: We plan to study the functions of PcrA which are critical for the growth and viability of S. aureus using genetic and biochemical approaches. The biochemical activities of various PcrA mutants will be correlated with their ability to support the growth of S. aureus and regulate RecA-mediated DNA recombination in vivo. Finally, we will study the possible roles of PcrA in UV DNA repair and the regulation of the SOS response in S. aureus.